Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with more than 200,000 new diagnoses each year. The oncogene Myc is a common driver of TNBC. However, despite important research into the biologic and molecular functions of Myc, no tractable therapies have emerged to inhibit this oncogenic transcription factor. TNBC and other Myc-driven cancers remain recalcitrant to current targeted therapies, underlining the urgent need to identify new vulnerabilities in these malignancies. In addition to promoting tumorigenesis, oncogenes such as Myc also produce unique stresses in cancer cells, collectively termed oncogenic stress. Using parallel whole-genome RNAi screens, we discovered that Myc imposes a new type of oncogenic stress on TNBCs that we term RNA splicing stress, or RSS (Kessler, Science 2012; Hsu, Nature 2015). In many cancer types, Myc (or other oncogenic insults) globally amplifies transcription leading to increased pre-mRNA burden on the spliceosome. Consequently, Myc-driven TNBCs are exquisitely sensitive to modest perturbations in the spliceosome (by enhancing RSS). Importantly, pharmacologic or genetic inhibitors of the spliceosome impair primary and metastatic progression of TNBC and are well tolerated in animal pre-clinical models. While spliceosome inhibitors have entered clinical trials, the mechanisms by which RSS and spliceosome inhibitors selectively kill cancer cells are unknown. Herein, we propose to discover the pathway(s) sensing and responding to RNA splicing stress and to delineate the mechanisms by which RSS triggers cancer cell death. Aim 1: Delineate the dsRNA-sensing pathways (DSP?s) stimulated by RSS in Myc+ TNBCs in vivo. Our preliminary data suggest that RSS induces TNBC apoptosis by triggering innate immune pathways that detect and respond to dsRNAs. By leveraging a TNBC PDX cohort with new genetic and proteomic platforms, we will determine how DSP?s and other stress response pathways are activated in response to spliceosome inhibition. Aim 2: Discover how RSS triggers dsRNA-sensing pathways in TNBCs. Many viral pathogens encode dsRNA genomes that are detected by dsRNA-sensors of the innate immune system. Our studies indicate RSS leads to cytoplasmic accumulation of intron-retained mRNAs, a potential source of dsRNAs. Using new sequencing and single-molecule microscopy methods, we will study the mechanisms by which RSS shapes the secondary structure and subcellular localization of TNBC transcriptomes and stimulates dsRNA-sensors. Aim 3: Delineate how DSPs and other stress response pathways govern TNBC response to spliceosome inhibitors. We hypothesize that there is a coordinated cellular response to RSS that parallels well-studied homeostatic stress responses like the unfolded protein response. By leveraging forward genetics and identifying new regulators of RSS, we propose to unveil a genetic framework for this novel type of cellular stress and provide new therapeutic inroads that exploit this cancer vulnerability.